Saccharomyces cerevisiae has been extensively studied as a host in biotechnological processes for the production of heterologous proteins of pharmaceutical interest. However, the productivity obtainable with this yeast species is low due to the lack of strong promoters and owing to the instability of transformed plasmid during a long-term culture under a nonselective condition. To deal with this instability problem, integrative transformation methods have been devised. For example, incorporation of multiple copies of genes encoding the heterologous proteins into specific sites in the chromosomal DNA was attempted by employing such targets as the transposable element Ty and ribosomal DNA plasmid. However, the copy number of the integrated genes resulting therefrom was observed to be low: that is, the aforementioned instability problem was left unresolved (Hinnen et al., "Gene Expression in Recombinant Yeast", in "Gene Expression in Microorganism", Smith, ed., Marcel Dekker, New York, 1995, p121).
A methylotrophic yeast, Hansenula polymorpha, on the other hand, possesses several strong promoters; and it is, therefore, an attractive cloning host for the production of heterologous proteins by way of multiple copy integration of heterologous genes into the chromosomal DNA thereof (Gleeson and Sudbery, Yeast, 4, 1(1988); Janowicz et al., Yeast, 7, 431(1991); Romanos et al., Yeast, 8, 423(1992); Faber et al., Yeast, 11, 1331(1995)). The strong promoters of Hansenula polymorpha include those associated with genes encoding methanol oxidase (MOX promoter), dihydroxyacetone synthase (DHAS promoter) and formate dehydrogenase (FMDH promoter). These promoters facilitate the expression of genes to the extent that the enzymes produced amount to 30 to 40% of the total cellular proteins (Gellissen et al., Trends Biotechnol., 10, 413(1992)). Moreover, it is known to be relatively easy to integrate multiple copies of heterologous genes into the chromosomal DNA of Hansenula polymorpha, thereby enhancing the stability of the heterologous gene expression during long-term culturing(Hoolenberg and Janowicz, EP 299,108 A; Janowicz et al., Nucleic Acid Res., 13, 3042(1985); Lederboer et al., Nuclei Acid Res., 13, 3063(1985)). Due to the above-mentioned advantages, there have been many studies on the production of recombinant proteins using Hansenula polymorpha as a cloning host.
When a transforming plasmid containing a DNA sequence for self-copying activity(i.e., autonomously replicating sequence, ARS) is introduced to Hansenula polymorpha, it remains in the episomal state only transiently after the transformation, the transformants with the episomal plasmid showing a very low miotic stability. However, when the transformants are subjected to a stabilization procedure, e.g., a series of selections of a marker gene of the transforming plasmid by employing a selective medium and then culturing the resultant in a nonselective medium for several generations, the transformed cells become stabilized through an integration of the transforming plasmid into the chromosomal DNA (Roggenkamp et al., Mol. Gen. Genet., 202, 302(1986)). During the process of such stabilization, certain cells acquire multiple copies of the heterologous gene in tandemly repeating gene units("multiple tandem repeats") integrated into the chromosomal DNA thereof.
The probability of obtaining cells having integrated multiple tandem genes increases with the self-copying ability of the ARS of the transforming plasmid (Roggenkamp et al., EP 0374282), but it is generally low. Thus, it is often required to go through time-consuming processes of selecting the cell having integrated multiple tandem genes; and, for this purpose, dominant selection markers have been used which confer varying degrees of resistance on the cell depending on the number of copies integrated therein.
Examples of the dominant selection markers mentioned above are: copper-resistant CUPI gene derived from S. cerevisiae (Fogel and Welch, Proc. Natl. Acad. Sci., U.S.A., 79, 5342(1982)), methotrexate-resistant dihydrofolate reductase gene derived from mouse cDNA (Zhu et al., Bio/Technol., 3, 451(1985)), G418-resistant aminoglycoside-3-phosphotransferase(APH) gene derived from Tn903 (Jamenez and Davies, Nature, 287, 869(1980)), hygromycin B-resistant hph gene derived from Streptomyces hygcroscopicus (Gritz and Davies, Gene, 25, 178(1983)), and sulfometuron methyl-resistant SMRI gene derived from S. cerevisiae (Casey et al., J. Ind. Brew., 94, 93(1988)). Among the dominant selection markers mentioned above, the APH gene has been most frequently used in various yeast hosts. However, because promoters originating from E. coli are not adequately functional in yeasts, the selection efficiency of the E. coli-derived APH gene is only about 10% of that achievable with an auxotrophic marker (Jimenes and Davies, Nature, 287, 869(1983); Webster and Dickson, Gene, 26, 243(1983)). The use of PGK promoter derived from S. cerevisiae in place of the E. coli-derived promotor raised the APH expression efficiency to a level equivalent to that of an auxotrophic marker (Hadfield et al., Curr. Genet., 18, 303(1990)). When the APH gene marker was attached to the MOX promoter and used in Hansenula polymorpha, the transformed cell was reported to have survived at a G418 concentration of 20 mg/ml(Gleeson and Sudbery, Yeast, 4, 1(1988)). It was also reported that the APH gene marker was tied to the promoter of alcohol dehydrogenase I(ADHI) of S. cerevisiae and used in a process to select cells containing multiple copies of heterologous genes (Janowicz et al., Yeast, 7, 431(1991)).
The MOX and FMDH promoters of Hansenula polymorpha are regulatory promoters whose expression is induced only when methanol is used as the carbon source (Gellisen et al., Trends Biotechnol., 10, 413(1992)). Accordingly, when these promoters are employed in conjunction with foreign genes, it is possible to control the cell culturing step separately from the step of expressing recombinant proteins. Although this feature is useful when the expressed recombinant protein inhibits the cell growth, it is more convenient to employ a strong constitutive promoter which can be induced by any carbon source. One such promoter is the promoter of glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene, which has been widely used in the expression of heterologous proteins in S. cerevisiae, Pichia pastoris and other yeasts (Kniskern et al., Gene, 46, 135(1986); Travis et al., J. Biol. Chem., 260, 4384(1985); Hallewell et al., Bio/Technol., 5, 363(1987); Rosenberg et al., Methods in Enzymol., 185, 341(1990); Waterham et al., Gene, 186, 37(1997)).
As to autonomously replicating sequences(ARS's) of yeasts, it is known that the chromosomal DNA of Saccharomyces cerevisiae contains 300 to 400 ARS's, each being separated by 30 to 40 kb (Newlon, C. S., Microbiol. Rev., 52, 568(1988)). Also, extensive studies have been conducted to characterize the ARS's of: S. cerevisiae (Foss et al., Science, 262, 2838(1993); Marahrens and Stillman, Science, 255, 817(1992); Rao et al., Mol Cell Biol., 14, 7643(1994); Rowley et al., Biochim. Biophys. Acta, 1217, 239(1994); Theis and Newlon, Mol Cell Biol., 14, 7652(1994)); Candida species (Cannon et al., Mol. Gen. Genet., 221, 210(1990); Sasnaukas et al., Yeast, 8, 253(1992); Yarrow lipolytica (Fournier et al., Proc. Natl. Acad. Sci., 90, 4912(1993); Matsuoka et al., Mol. Gen. Genet., 237, 327(1993)); Schizosaccharomyces pombe (Caddle and Calos, Mol. Gen. Genet., 14, 1796(1993); and Pichia species (Phillips Petroleum Co., U.S. Pat. No. 4,837,148).
There are two representative strains of Hansenula polymorpha: CBS4732(ATCC 34438, Hazeu et al., Arch. Microbiol., 87, 185(1972)) and DL-1(ATCC 26012, Levine and Cooney, Appl. Microbiol., 26, 982(1973)). The strain CBS4732 has been employed in the production of recombinant proteins; and several ARS's of Hansenula polymorpha(HARS's) of strain CBS4732 have been cloned (Roggenkamp et al., Mol. Gen. Genet., 202, 302(1986); Bogdanova et al., Yeast, 11, 343(1995)). However, no studies have so far been conducted concerning the biochemical specificity of HARS's. The DL-1 strain, on the other hand, has never been utilized in a recombinant protein synthesis despite the advantages it offers over the CBS4732 strain: unlike CBS4732, it is thermotolerant at a growth temperature ranging from 37 to 50.degree. C., capable of growing at a high methanol concentration due to its high methanol oxidase activity, and gives a higher protein yield in a comparative study wherein vectors containing the respective MOX promoters of the two strains are used (Sohn et al., Appl. Microbiol. Biotechnol., 3, 65(1993). The MOX promoters of the two strains were found to have some differences at the sequence related to the methanol-induced expression (Godecke et al., Gene, 139, 35(1995)), and the homologous recombination efficiency was observed to be much higher with DL-1 than with CBS4732. Accordingly, the use of strain DL-1 as a cloning host in the production of recombinant proteins is expected to offer the following advantages over the case of using strain CBS4732.
First, because strain DL-1 grows fast in a medium having a high methanol concentration, precise control of the methanol concentration is not required for the induced expression of the heterologous gene. This characteristic property is particularly suitable when the production of the heterologous protein is growth-dependent. Second, as DL-1 grows well in a medium containing only methanol as the carbon source, it is presumed that the activity of DL-1 MOX promoter in the expression of the heterologous gene is higher than that of CBS4732. Third, DL-1 exhibits a high homologous recombination efficiency, which facilitates the integration of heterologous genes thereinto.
However, in order to employ Hansenula polymorpha DL-1 as the host in the production of recombinant proteins, it is necessary to find HARS's having high self-copying activities and also to establish an efficient method for selecting H. polymorpha cells carrying tandemly integrated foreign genes encoding said proteins.